Diagnostic array for aseptic encephalitis

描述(由申请人提供)： 摘要无菌性脑炎和脑膜炎是一种潜在的致命性疾病，定义为急性脑肿胀或覆盖在脑和脊髓上的保护膜。这些疾病可由病毒、细菌、真菌和寄生虫引起。病毒是脑炎最常见的原因，在所有无菌性脑炎中，有85%是由肠道病毒引起的。人类疱疹病毒占所有病毒感染的4%，西尼罗河病毒是美国最常见的虫媒病毒感染。根据疾控中心的数据，大多数人类肠道病毒性脑炎病例都没有并发症。另一方面，如果不治疗，单纯疱疹病毒性脑炎会导致大约70%的病例迅速死亡。脑膜炎奈瑟氏菌可通过迅速扩散的点状皮疹与其他原因的脑膜炎区分开来，而其他细菌性和真菌性脑膜炎/脑炎可通过脑脊液的生化和革兰氏染色与病毒原因区分开来。重要的是，脑脊液感染引起的并发症和适当的治疗策略取决于感染的类型和涉及的种类。因此，对于疾病的无菌原因，病例适应治疗依赖于快速诊断和区分病毒感染。十多年来，核酸扩增和检测分析一直被认为是脑脊液感染的首选测试，但目前只有三种FDA批准的病毒诊断测试，每种测试一次只能检测到一种病毒。因此，本项目的目的是开发和试验一种用于脑炎和脑膜炎诊断的“闭合扩增”凝胶元件RT-PCR阵列。该检测是基于沃兹沃斯中心病毒疾病实验室开发的一套实时聚合酶链式反应和逆转录酶聚合酶链式反应检测方法。第一阶段的性能目标是一个封闭的扩增子RT-PCR阵列，用于检测肠道病毒、疱疹病毒(HSV-1、HSV-2、VZV、CMV、HHV-6)和西尼罗河病毒，每次反应的分析LOD为50个基因组拷贝。该分析将包含一个外源添加、定量提取和扩增对照，在相同的多重反应中进行扩增和检测。在第一阶段成功完成任何一个特定目标将保证第二阶段的建议，重点是试剂的稳定性、包装和保质期；临床前实验以证明分析的重复性、重复性、敏感性和特异性；以及开发用于分析数据分析和报告的自动化算法和决策规则。 公共卫生相关性：闭合扩增凝胶元件微阵列将是首个将RT-PCR和微阵列技术结合在一起的简单、廉价的无菌脑和脑膜诊断试剂盒。本文开发的底层平台同样将在传染病诊断的许多领域得到广泛应用。

DESCRIPTION (provided by applicant): ABSTRACT Aseptic encephalitis and meningitis are potentially fatal diseases defined by acute swelling of the brain or protective membranes covering the brain and spinal cord. These diseases can be caused by viruses, bacteria, fungi and parasites. Viruses are the most common cause of encephalitis, with >85% of all aseptic encephalitis caused by enterovirus. Human herpes viruses account for 4% of all viral infections, and West Nile virus is the most common of the arboviral infections in the U.S. According to the CDC, most cases of human enteroviral encephalitis resolve without complication. One the other hand, herpes simplex encephalitis results in rapid death in approximately 70% of cases if left untreated. Neisseria meningitides can be differentiated from other causes of meningitis by a rapidly spreading petechial rash, and other bacterial and fungal meningitis/encephalitis can readily be differentiated from viral causes by biochemistry and gram staining of the cerebrospinal fluid. Importantly, complications arising from CSF infection and appropriate treatment strategies depend on the type of infection and the species involved. For aseptic causes of the disease, case-adapted therapy is therefore dependent upon rapidly diagnosing and differentiating between viral infections. Nucleic acid amplification and detection assays have been considered the test of choice for CSF infections for more than a decade, yet there are only three FDA-approved viral diagnostic tests, each of which only detects one virus at a time. The objective of this project is therefore to develop and trial a "closed amplicon" gel element RT-PCR array for the diagnosis of encephalitis and meningitis. The assay is based on a set of real- time PCR and reverse transcriptase PCR assays developed by the Laboratory of Viral Diseases at the Wadsworth Center. The performance goal for Phase 1 is a closed amplicon RT-PCR array for the detection of enterovirus, herpes viruses (HSV-1, HSV-2, VZV, CMV, HHV-6), and West Nile virus, with an analytical LoD of d 50 genome copies per reaction. The assay will contain an exogenously spiked, quantified extraction and amplification control which is amplified and detected in the same multiplex reaction. Successful completion of either Specific Aim in Phase 1 will warrant a Phase 2 proposal focused on reagent stability, packaging and shelf life; pre-clinical experiments to demonstrate assay reproducibility, repeatability, sensitivity and specificity; and developing automated algorithms and decision rules for assay data analysis and reporting. PUBLIC HEALTH RELEVANCE: The closed-amplicon gel element microarray will be the first-of-its-kind combination of RT-PCR and microarray technology in a simple, inexpensive test kit for the diagnosis of aseptic encephalities and meningits. The underlying platform developed herein will likewise find broad application in many areas of infectious disease diagnostics.